Project Summary The fungal pathogen Cryptococcus neoformans is estimated to cause disease in more than 1,000,000 people worldwide every year, resulting in greater than 600,000 deaths. However, not all Cryptococcus isolates are killers; some are relatively benign while oth- ers are hypervirulent. Differences in pathogenicity are often correlated with variation in key morphological and physiological features such as the ability to grow at high tem- peratures, the size of the protective polysaccharide capsule surrounding the yeast cell, or resistance to antifungal drugs. In order to develop better ways to prevent and treat cryptococcal disease we need to understand the underlying genetic differences that lead to changes in virulence and virulence-related traits. We also need to understand how frequently and how wide spread these genetic differences are in different parts of the world. To tackle these problems, the proposed research will use a combination of experi- mental and statistical approaches to dissect the causal genetic basis of variation in vir- ulence and virulence-related traits. In Aim 1 the investigators will employ a statistical technique called Quantitative Trait Locus (QTL) mapping, that exploits genotypic and phenotypic differences among offspring derived from genetic crosses to identify regions of the genome (loci) and DNA changes (alleles) that contribute to differences in virulence traits. The investigators will validate the contributions of the loci identi?ed in this man- ner using gene deletions and related techniques. In Aim 2 the investigators will subject genetically diverse populations of Cryptococcus to selection in animal hosts. Following selection, pooled genome sequencing will be used to identify loci and alleles that are favoring during infection. This technique provides an unbiased approach for discover- ing loci that contribute to virulence, and will both complement and extend the results of Aim 1. In Aim 3 the investigators will put their ?ndings in the context of natural populations of Cryptococcus by studying the frequency and geographic distributions of virulence alleles identi?ed in Aims 1 and 2. This aim will employ a large, world-wide sample of Cryptococcus strains isolated both from clinical settings and from the natural environment. The investigators will use this information to identify regions of the world where there are high frequencies of virulent genotypes, or where there is potential for in- creased virulence through processes like recombination or hybridization. 1